CN106324173B - A kind of measure screening technique of the laryngocarcinoma serum difference metabolin based on reverse-phase chromatography flight time mass spectrum - Google Patents

Abstract

A method for the determination and screening of differential metabolites in laryngeal cancer serum based on reversed-phase chromatography time-of-flight mass spectrometry, characterized in that: after the serum is precipitated with an organic solvent-methanol, it is then centrifuged, vacuum centrifuged and dried, and then redissolved in 200 µL of pure Water was introduced into reversed-phase chromatography time-of-flight mass spectrometer for determination. After peak alignment, correction and standardization, multivariate statistical analysis and spectral library search, differential metabolites in the serum of laryngeal cancer volunteers and healthy volunteers could be found. The present invention is a brand-new identification method of differential metabolites in serum, which can reflect the changes of serum metabolites in patients with laryngeal cancer, and will help the diagnosis, clinical treatment, prognosis judgment and research of pathogenesis of laryngeal cancer .

Description

Detection of differential metabolites in laryngeal cancer serum based on reversed-phase chromatography time-of-flight mass spectrometry custom screening method

Technical field:

The invention belongs to the technical field of physical and chemical testing of serum, in particular to a method for measuring and screening differential metabolites in serum of laryngeal cancer based on reversed-phase chromatography time-of-flight mass spectrometry.

Background technique:

The common type of laryngeal cancer is squamous cell carcinoma, which has the characteristics of high incidence and high recurrence rate, and is prone to cervical lymph node metastasis. In the treatment of laryngeal cancer, laryngectomy is the main method of surgery, supplemented by radiotherapy and chemotherapy, but it is not sensitive to chemotherapy, and it is easy to cause problems such as poor pronunciation and swallowing function of patients, and reduced quality of life. The treatment effect of early laryngeal cancer The curative effect of advanced laryngeal cancer is better, but there is still a lack of effective biomarkers for the early diagnosis of laryngeal cancer. Therefore, the discovery of new biomarkers is of great significance for the early diagnosis and treatment of laryngeal cancer. Metabolomics is a newly developed discipline following genomics and proteomics. It is a new discipline that simultaneously conducts qualitative and quantitative analysis of all low-molecular-weight metabolites of a certain organism or cell in a specific physiological period. Metabolomics technology can discover new biomarkers, which will help the diagnosis, clinical treatment, prognosis and pathogenesis of head and neck cancer.

For the discovery of biomarkers with diagnostic functions, non-invasive or less invasive body fluids, such as blood and urine, are often used. Urine contains many metabolic end products, as well as metabolites of nutrients, drugs and exogenous substances, while blood often responds to some endogenous metabolites. However, for clinical diagnosis, blood is often used, because the collection of 24-hour urine samples is cumbersome and error-prone, and the concentration of compounds in morning urine is easily affected by the liquids (such as beverages, water, etc.) that the subjects ingest. The impact cannot fully represent the subject's state at that time, but the blood flowing through the subject's body can well reflect the subject's physical condition. At present, serum has been widely used in the study of differential metabolites of lung cancer and nasopharyngeal carcinoma, but the study of differential metabolites of laryngeal cancer has not been reported.

Invention content:

The purpose of the present invention is to overcome the defects of the prior art and establish a simple, fast and selective reversed-phase chromatography time-of-flight mass spectrometry method for screening differential metabolites in the serum of laryngeal cancer volunteers and healthy volunteers. , Accurate identification of differential metabolites in serum.

The purpose of the present invention is achieved through the following technical solutions:

A method for the determination and screening of differential metabolites in laryngeal cancer serum based on reversed-phase chromatography time-of-flight mass spectrometry, which uses the serum of laryngeal cancer volunteers and healthy volunteers as samples for measurement, comparison, and screening, specifically including the following process steps:

a. Thaw the blood at room temperature, pipette 400 µL of serum, add 1200 µL of methanol, vortex mix for 15 s, and then centrifuge at 15800 g for 15 min in a centrifuge;

b. Pipette 370 µL of the supernatant into a 2 mL centrifuge tube, then dry it in a vacuum centrifuge, redissolve it in 200 µL of pure water, and put it into a chromatographic bottle for testing;

c. Reverse-phase chromatography time-of-flight mass spectrometry analyzes all serum samples one by one to obtain raw data;

d. After converting the original data into the mzXML format, after performing peak alignment, correction, and noise filtering on the data, SIMCA-P was used for multivariate statistical analysis, and metabolites with VIP greater than 1 were screened.

e. T-test with SPSS, for P <0.05 and VIP greater than 1, it is a potential differential metabolite.

f. For potential differential metabolites, use methods such as spectral library search or standard comparison for verification, which are differential metabolites.

In step c, all samples were separated by ACQUITY UPLC ^® BEH column (2.1 × 150 mm, 1.7 µm) produced by Water Company, the column temperature was 50 ºC, mobile phase A was 0.1% formic acid water, mobile phase B 0.1% formic acid acetonitrile, mobile phase flow rate 385 μL/min, gradient elution conditions: 0-1 min, 5%B-5%B; 1-10 min, 5%B-20%B; 10-12 min, 20%B-60%B; 12-33 min, 60%B-100%B; 33-35 min, 100%B-100%B; 35-36 min, 100%B-5%B, and It was maintained for 2 minutes, the total analysis time was 38 minutes, and the injection volume was 5 μL.

In step c, the conditions of the time-of-flight mass spectrometry detector: electrospray ionization source, multiple reaction monitoring positive ion scanning mode; the spray voltage is 3500 V, the drying gas is 4 L/min (180°C), and the nebulizing gas is 0.04 MPa , with a collision energy of 7eV and a transit time of 100 µs.

Stability and repeatability of the inventive method:

Take an equal amount of serum from each serum sample of laryngeal cancer volunteers and healthy volunteers, and mix it evenly, which is the quality control (QC) sample. The quality control (QC) sample is used to evaluate the stability and repeatability of the instrument. To ensure the reliability of the experimental results, select m/z as 265.2024, 318.2977, 496.3361, 675.6710 and 782.5508 to evaluate the stability and repeatability of the instrument, and the relative standard deviation (RSD) of the retention time and peak area of these five endogenous compounds See Table 1, the RSD of the retention time is less than 1%, and the RSD of the peak area is less than 15%, indicating that the stability and repeatability of the system meet the requirements

Table 1 Stability and repeatability of the method

Number of peaks m/z Retention time RSD (%) Peak area RSD (%) 1 265.2024 0.84 9.26 2 318.2977 0.65 5.76 3 496.3361 0.28 9.74 4 675.6710 0.29 12.4 5 782.5508 0.19 7.55

The method of the present invention is a new method for discovering differential metabolites in laryngeal cancer serum, and optimizes relevant detection conditions of reversed-phase chromatography time-of-flight mass spectrometry, mainly optimizing ion source conditions, chromatographic columns and mobile phase systems. The inventive method has following good effect:

①Liquid chromatography time-of-flight mass spectrometry has higher sensitivity.

② This method is simple in pretreatment, does not need to go through a complicated solid phase extraction process, can improve the analysis throughput, and has the advantages of rapidity and good repeatability.

Description of drawings

Figure 1. Typical base peak chromatograms (the upper picture is for healthy volunteers, and the lower picture is for laryngeal cancer volunteers);

Figure 2. Score plots of serum samples from laryngeal cancer volunteers and healthy volunteers, 1 is healthy and 2 is laryngeal cancer patients.

Detailed ways

The present invention will be further described below in conjunction with examples, but not limit the present invention.

A method for the determination and screening of differential metabolites in laryngeal cancer serum based on reversed-phase chromatography time-of-flight mass spectrometry. The test process is that the serum undergoes organic solvent-methanol precipitation of proteins, then centrifuges, vacuum centrifuges and dries, and then redissolves in 200 µL of pure Water was introduced into ultra-high performance liquid chromatography-electrospray ionization source-time-of-flight mass spectrometer for determination.

Example 1:

1. Instruments and reagents: The liquid chromatograph used in the experiment is Dionex’s UltiMate 3000, including liquid phase pump (HPG-3400 SD), autosampler (WPS-3000 SL) and column thermostat (TCC-3000 SD), All samples were separated by ACQUITY UPLC ^® BEH column (2.1 × 150 mm, 1.7 µm) produced by Water Company, and the column temperature was 50 ºC.

Chromatographic grade formic acid, with a purity of 98%, was purchased from TEDIA; acetonitrile, with a purity of 99.9%, was purchased from DUKSAN PURE CHEMIVALS, Korea; 1-hexadecanoyl-sn-glycerol-phosphocholine , with a purity of more than 99%, purchased from Sigma-Aldrich; the experimental water was deionized water.

2. Sample processing: thaw serum at room temperature, pipette 400 µL of serum, add 1200 µL of methanol, vortex mix for 15 s, then centrifuge in a centrifuge at 15800 g for 15 min, pipette 370 µL of supernatant in 2 mL centrifuge tube, then dried by centrifugation in a vacuum centrifuge, then redissolved in 200 µL of pure water, and put into a chromatographic vial for testing.

3. Determination method: draw 5 µL of pretreated serum sample and inject it into reversed-phase chromatography time-of-flight mass spectrometry system for separation and analysis.

4. After the original data is converted to mzXML format, peak identification, extraction and retention time correction are performed on the data. The specific parameter settings are: the allowable error of mass-to-charge ratio (m/z) is 10 ppm, and the width of the chromatographic peak is 10 ppm. -60 s, and the step size during retention is set to 1 m/z.

5. Then use SIMCA-P 13.0 Demo software to normalize the data extracted from XCMS online, and perform principal component analysis, partial least squares discriminant analysis or orthogonal partial least squares discriminant analysis (OPLS-DA), according to OPLS The results of -DA found metabolites with relatively large changes, and then used SPSS 20.0 to perform T-test.

6. According to VIP>1 and P<0.05, more than 100 potential differential metabolites were found, and then the time-of-flight mass spectrometer was used for secondary fragmentation scanning in MRM or AutoMS ⁿ mode, and the allowable error range was 10 ppm. Spectral library searches were performed using secondary fragments under certain conditions.

Example 2: According to the method described in Example 1, 22 cases of laryngeal cancer volunteers and 26 cases of healthy volunteers were selected for serum samples, and one of the differential metabolites was identified as 1-hexadecanoyl-sn-glycerol-phosphate choline.

Claims (1)

1. a kind of measure screening technique of the laryngocarcinoma serum difference metabolin based on reverse-phase chromatography flight time mass spectrum, feature exist In：It is to be measured, compared as sample using laryngocarcinoma volunteer and healthy volunteer's serum, screened, specifically includes following technique step Suddenly：

A, blood thaws at room temperature, pipettes the serum of 400 μ L, adds in the methanol of 1200 μ L, 15 s of vortex mixed, then with 15800 g centrifugal force centrifuge 15 min in centrifuge；

B, the supernatant of 370 μ L is pipetted in the centrifuge tube of 2 mL, and then the centrifugal drying in vacuum centrifuge, then redissolves In the pure water of 200 μ L, it is then charged into be measured in chromatogram bottle；

C, reverse-phase chromatography flight time mass spectrum analyzes all serum samples one by one, obtains initial data；

Reverse-phase chromatography condition is：Using ACQUITY UPLC^®BEH columns, specification 2.1 × 150 mm, 1.7 μm points From column temperature is 50 oC, and mobile phase A is 0.1% formic acid water, and Mobile phase B is 0.1% formic acid acetonitrile, and flow rate of mobile phase is 385 μ L/min, gradient elution, bulk analysis time are 38 min, and sample size is 5 μ L；Condition of gradient elution is specially：0-1 Min, 5%B-5%B；1-10 min, 5%B-20%B；10-12 min, 20%B-60%B；12-33 min, 60%B-100%B；33-35 Min, 100%B-100%B；35-36 min, 100%B-5%B, and maintain 2 min；

The condition of flight time mass spectrum detector：Electric spray ion source, multiple-reaction monitoring cation scan mode；Spray voltage is 3500 V, dry gas are 4 L/min, and atomization gas is 0.04 MPa, and collision energy is 7 eV, and transmission time is 100 μ s；

D, after converting raw data into the form of mzXML, after making an uproar to data progress peak alignment, correction, filter, using SIMCA-P Multivariate data analysis software carries out multivariate statistical analysis, and screening VIP is more than 1 metabolin；

E, T inspections are carried out with SPSS, forP<0.05 and VIP is potential difference metabolin more than 1；

F, for potential difference metabolin, verified using library searching or standard items comparison method, be difference metabolism Object.